At the outset several terms should be defined. "Cellular matter" or "cell" refers to a living structure, composed of a mass of protoplasm, enclosed in a membrane and containing a nucleus. It may or may not be part of a larger structure. "Tissue" means a collection of similar cells and the intercellular substances surrounding them. There are four basic tissues in the human body: (1) epithelium; (2) connective tissues, including blood, bone and cartilage; (3) muscle tissue; and (4) nerve tissue. "Cryoprotectant" refers to chemical compounds which are added to biological samples in order to minimize the deleterious effects of cryopreservation procedures. "Osmotic effects" refers to the alteration in the osmotic strength of the suspending media caused by conversion of water to ice or ice to water. This conversion results in substantial flow of water across membranes of unfrozen cells, causing volume changes during freezing and thawing. "Viability" refers to the ability of frozen and thawed cells to perform their normal functions. Viability is usually expressed as the ability of the cells to reproduce, metabolize, exclude vital dyes or carry out some other metabolic function. The viability of the frozen and thawed samples should always be compared to the ability of unfrozen cells obtained at the same time to carry out the same function.
There has been an increase in recent years of person willing to donate various organs for transplantation or research purposes. Along with this national networks have been created to match available organs with needy recipients, resulting in a growing need to store organs for extended periods of time. In 1987 there will have been approximately 400,000 transplantation and implantation procedures, involving such tissues as heart valve, cornea, pancreas, skin, blood vessel, tendon/ligament, bone, bone marrow, nerve and others. The major advances in transplantation of tissues are occurring because of efficient sterile procurement techniques, recognition of a group of immunologically privileged tissues, development of techniques for the avoidance of the transplant recipient's immune surveillance system, and improvements in tissue preservation methods. Storage of this tissue has become more important to enable physicians to procure and match tissues with recipients.
Historically, several approaches for tissue storage have been used. The most commonly used and promising method has been cryopreservation. Some alternative method has been cryopreservation. Some alternative methods have been freeze drying, chemical treatment, tissue culture prior to transplantation, and storage at refrigeration temperatures. Storage at refrigeration temperatures is acceptable for short periods of time, but can result in reduced viability of the tissue if stored too long. Freezing protocols must be designed to optimize tissue viability. These methods, if not properly controlled, can lead to cell damage.
Two major mechanism for injury to cells and tissues during freezing have been emphasized. First, there are the obvious mechanical injuries which can occur due to either extra or intracellular ice crystal formation. Second, there is the danger of osmotic dehydration. Current cryopreservation technology consists of trying to maintain a balance between these two forms of injury. Basically, when freezing is performed at a rapid rate, there is a tendency for ice crystals to form both intracellularly and extracellularly. However, when cryopreservation is performed at slower rates, there is a tendency for ice crystal formation to occur first in the extracellular medium. As the extracellular ice forms, the cells are exposed to an increasingly hyperosmotic environment. This is due to water sequestration as the ice crystals grow. The cells shrink due to transport of water out of the cell in response to the osmotic imbalance caused by the increasing extracellular solute concentrations. The net result of combining optimal cooling rates and cryoprotective agents is that less of the freezable intracellular water will be converted to ice and osmotic cellular dehydration is limited.
The field of cryopreservation dates from 1949, when Polge, Smith, and Parkes discovered the protective properties of glycerol for bull sperm [Nature 164;666 (1949)] Subsequently, in 1959, Lovelock and Bishop [Nature 183:1394 (1959)] reported the protective activities of dimethysulfoxide ("DMSO") is preventing freezing injury to living cells. DMSO and glycerol have since become the most widely utilized cryoprotectant.
Permeating cryoprotective agents, such as DMSO and glycerol, act by penetrating the cell membrane and reducing the intracellular water concentration, thereby reducing the amount of ice formed at any temperature.
There are a variety of so-called nonpermeating protective agents. These agents include such compounds as polyvinylpyrrolidone, hydroxyethyl starch, monosaccharides, and sugar alcohols. In addition, both permeating and nonpermeating cryoprotectants act directly on the cell membranes. The mechanism of the nonpermeating cryoprotectants is not clear, but may involve changes in colloidal osmotic pressure and modifications of the behavior of membrane associated with water by ionic interaction. For some cells, combination of these two classes of cryoprotective agents may give optimal viability.
There is a need, then, for an agent that can overcome the problems of ice crystal formation, toxicity and osmotic dehydration possible during the cryopreservation process. Such an agent would be nontoxic to the tissue or cell, inexpensive to obtain, and convenient to use.